Acute and chronic ethanol (EtOH) administration or consumption markedly decreases total tissue Mg2+ content in humans and experimental animals. The results obtained during the previous period of funding provide a major progress in understanding how Mg2+ depletion occurs. We have shown that EtOH induces Mg2+ loss through at least two mechanisms: 1) by decreasing cytosolic ATP content, EtOH reduces Mg2+ buffering in the cytoplasm, which results in a dose- and time-dependent extrusion of Mg2+ from the cell via a Na+-dependent mechanism, and 2) by inhibiting Mg2+ accumulation, EtOH prevents the cell from restoring normal Mg2+ level. These effects are evident following acute administration of EtOH and become more pronounced after prolonged alcohol consumption in the diet. The goal of the present project is to characterize, at the cellular and sub-cellular level, the detailed mechanisms by which acute and chronic EtOH administration impairs Mg2+ homeostasis and transport mechanisms in liver cells, and the short- and long-term implications that Mg2+ loss has for specific cellular functions. It has been suggested that a persistent decrease in Mg2+ content is deleterious to the tissue in which it occurs, leading to the impairment of metabolic functions within the cell (e.g. collagen deposition) and ultimately to alcohol-related pathologies such as hepatitis and cirrhosis. Hence, determining the mechanisms involved in Mg2+ depletion will permit to operate preventively to reduce the insurgence of these pathologies in liver cells. This project has three principal aims: 1) To test the hypothesis that ethanol prevents Mg2+ accumulation by specifically inhibiting the protein kinase C signaling pathway that activates the Mg2+ entry mechanism in liver cell membrane, 2) To determine the effect of the Mg2+ loss induced by ethanol on mitochondria bioenergetics and endoplasmic reticulum Ca2+ handling, and 3) To determine whether the Mg2+ loss can be reverted after removal of alcohol from the diet, or can be prevented by a Mg2+-enriched diet. The results of these studies will provide the necessary background for future studies aimed to correlate mechanistically Mg2+ deficiency with impairment of body or organ functions, and provide means to prevent and/or correct alcohol-induced, Mg2+-mediated functional deficiencies within tissues.